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Background and Purpose: Although isoprenaline (ISO) is known to activate several ion currents in mammalian myocardium, little is known about the role of action potential morphology in the ISO-induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca²⁺ current (I(Ca)), slow delayed rectifier K⁺ current (I(Ks)) and fast delayed rectifier K⁺ current (I(Kr)) were studied and compared in a frequency-dependent manner using canine isolated ventricular myocytes from various transmural locations.Experimental Approach: Action potentials were recorded with conventional sharp microelectrodes; ion currents were measured using conventional and action potential voltage clamp techniques.Key Results: In myocytes displaying a spike-and-dome action potential configuration (epicardial and midmyocardial cells), ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. ISO-induced action potential shortening was absent in endocardial cells and in myocytes pretreated with 4-aminopyridine. Application of the I(Kr) blocker E-4031 failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of the I(Ks) blocker HMR-1556. Both action potential shortening and elevation of the plateau were prevented by pretreatment with the I(Ca) blocker nisoldipine. Action potential voltage clamp experiments revealed a prominent slowly inactivating I(Ca) followed by a rise in I(Ks) , both currents increased with increasing the cycle length.Conclusions and Implications: The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of I(Ks) - but not I(Kr) - may be responsible for the observed shortening of action potentials. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Although isoprenaline (ISO) is known to activate several ion currents in mammalian myocardium, little is known about the role of action potential morphology in the ISO-induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca2+ current ( ICa), slow delayed rectifier K+ current ( IKs) and fast delayed rectifier K+ current ( IKr) were studied and compared in a frequency-dependent manner using canine isolated ventricular myocytes from various transmural locations. EXPERIMENTAL APPROACH Action potentials were recorded with conventional sharp microelectrodes; ion currents were measured using conventional and action potential voltage clamp techniques. KEY RESULTS In myocytes displaying a spike-and-dome action potential configuration (epicardial and midmyocardial cells), ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. ISO-induced action potential shortening was absent in endocardial cells and in myocytes pretreated with 4-aminopyridine. Application of the IKr blocker E-4031 failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of the IKs blocker HMR-1556. Both action potential shortening and elevation of the plateau were prevented by pretreatment with the ICa blocker nisoldipine. Action potential voltage clamp experiments revealed a prominent slowly inactivating ICa followed by a rise in IKs, both currents increased with increasing the cycle length. CONCLUSIONS AND IMPLICATIONS The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of IKs- but not IKr- may be responsible for the observed shortening of action potentials. [ABSTRACT FROM AUTHOR]

BACKGROUND: Conventional microelectrode techniques were used to compare the concentration-dependent effects of vesnarinone (0.1-100 µM) and amrinone (1 µM-1 mM) on action potential duration (APD) and developed force in both isolated dog ventricular trabeculae and Purkinje strands. METHODS AND RESULTS: Both drugs increased contractility of trabecular muscle preparations, while, in Purkinje strands, vesnarinone failed to increase developed force during continuous pacing at 2 Hz. Vesnarinone lengthened APD in both preparations; although this effect was more marked in Purkinje strands. Ventricular muscle APD was not affected by amrinone (1 µM to 1 mM), while, in Purkinje strands, amrinone produced a biphasic effect on APD. Low concentrations (1-100 µM) of amrinone shortened Purkinje fiber APD, while only the highest concentration (1 mM) used lengthened APD. In addition, in Purkinje strand preparations the effects of vesnarinone (10 µM) on APD and developed force were proportional to pacing cycle length at frequencies slower than 2 Hz; however, at frequencies faster than 2 Hz vesnarinone decreased developed force while APD was lengthened. In ventricular trabecular muscle preparations, the effects of vesnarinone were not affected by frequency. CONCLUSIONS: These results indicate clear differences between the effects of vesnarinone and amrinone in isolated cardiac preparations. These differences in experimental effects in isolated cardiac preparations may help provide an explanation for the disappointing clinical response of patients in heart failure to amrinone, while vesnarinone has appeared to be beneficial. [ABSTRACT FROM AUTHOR]

Current density, the membrane current value divided by membrane capacitance (C m ), is widely used in cellular electrophysiology. Comparing current densities obtained in different cell populations assume that C m and ion current magnitudes are linearly related, however data is scarce about this in cardiomyocytes. Therefore, we statistically analyzed the distributions, and the relationship between parameters of canine cardiac ion currents and C m , and tested if dividing original parameters with C m had any effect. Under conventional voltage clamp conditions, correlations were high for I K1 , moderate for I Kr and I Ca,L , while negligible for I Ks . Correlation between I to1 peak amplitude and C m was negligible when analyzing all cells together, however, the analysis showed high correlations when cells of subepicardial, subendocardial or midmyocardial origin were analyzed separately. In action potential voltage clamp experiments I K1, I Kr and I Ca,L parameters showed high correlations with C m . For I NCX , I Na,late and I Ks there were low-to-moderate correlations between C m and these current parameters. Dividing the original current parameters with C m reduced both the coefficient of variation, and the deviation from normal distribution. The level of correlation between ion currents and C m varies depending on the ion current studied. This must be considered when evaluating ion current densities in cardiac cells., (© 2024. The Author(s).)

Slow inward currents (SICs) are known as excitatory events of neurons elicited by astrocytic glutamate via activation of extrasynaptic NMDA receptors. By using slice electrophysiology, we tried to provide evidence that SICs can elicit synaptic plasticity. Age dependence of SICs and their impact on synaptic plasticity was also investigated in both on murine and human cortical slices. It was found that SICs can induce a moderate synaptic plasticity, with features similar to spike timing-dependent plasticity. Overall SIC activity showed a clear decline with aging in humans and completely disappeared above a cutoff age. In conclusion, while SICs contribute to a form of astrocyte-dependent synaptic plasticity both in mice and humans, this plasticity is differentially affected by aging. Thus, SICs are likely to play an important role in age-dependent physiological and pathological alterations of synaptic plasticity., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

The present study was designed to test the hypothesis that the selectivity of blocking the late Na + current (I NaL ) over the peak Na + current (I NaP ) is related to the fast offset kinetics of the Na + channel inhibitor. Therefore, the effects of 1 µM GS967 (I NaL inhibitor), 20 µM mexiletine (I/B antiarrhythmic) and 10 µM quinidine (I/A antiarrhythmic) on I NaL and I NaP were compared in canine ventricular myocardium. I NaP was estimated as the maximum velocity of action potential upstroke (V + max ). Equal amounts of I NaL were dissected by the applied drug concentrations under APVC conditions. The inhibition of I NaL by mexiletine and quinidine was comparable under a conventional voltage clamp, while both were smaller than the inhibitory effect of GS967. Under steady-state conditions, the V + max block at the physiological cycle length of 700 ms was 2.3% for GS967, 11.4% for mexiletine and 26.2% for quinidine. The respective offset time constants were 110 ± 6 ms, 456 ± 284 ms and 7.2 ± 0.9 s. These results reveal an inverse relationship between the offset time constant and the selectivity of I NaL over I NaP inhibition without any influence of the onset rate constant. It is concluded that the selective inhibition of I NaL over I NaP is related to the fast offset kinetics of the Na + channel inhibitor.

Late sodium current (I Na,late ) is an important inward current contributing to the plateau phase of the action potential (AP) in the mammalian heart. Although I Na,late is considered as a possible target for antiarrhythmic agents, several aspects of this current remained hidden. In this work, the profile of I Na,late , together with the respective conductance changes (G Na,late ), were studied and compared in rabbit, canine, and guinea pig ventricular myocytes using the action potential voltage clamp (APVC) technique. In canine and rabbit myocytes, the density of I Na,late was relatively stable during the plateau and decreased only along terminal repolarization of the AP, while G Na,late decreased monotonically. In contrast, I Na,late increased monotonically, while G Na,late remained largely unchanged during the AP in guinea pig. The estimated slow inactivation of Na + channels was much slower in guinea pig than in canine or rabbit myocytes. The characteristics of canine I Na,late and G Na,late were not altered by using command APs recorded from rabbit or guinea pig myocytes, indicating that the different shapes of the current profiles are related to genuine interspecies differences in the gating of I Na,late . Both I Na,late and G Na,late decreased in canine myocytes when the intracellular Ca 2+ concentration was reduced either by the extracellular application of 1 µM nisoldipine or by the intracellular application of BAPTA. Finally, a comparison of the I Na,late and G Na,late profiles induced by the toxin of Anemonia sulcata (ATX-II) in canine and guinea pig myocytes revealed profound differences between the two species: in dog, the ATX-II induced I Na,late and G Na,late showed kinetics similar to those observed with the native current, while in guinea pig, the ATX-II induced G Na,late increased during the AP. Our results show that there are notable interspecies differences in the gating kinetics of I Na,late that cannot be explained by differences in AP morphology. These differences must be considered when interpreting the I Na,late results obtained in guinea pig.

Aims: Heart failure with reduced ejection fraction (HFrEF) is a disease with high mortality and morbidity. Recent positive inotropic drug developments focused on cardiac myofilaments, that is, direct activators of the myosin molecule and Ca 2+ sensitizers for patients with advanced HFrEF. Omecamtiv mecarbil (OM) is the first direct myosin activator with promising results in clinical studies. Here, we aimed to elucidate the cellular mechanisms of the positive inotropic effect of OM in a comparative in vitro investigation where Ca 2+ -sensitizing positive inotropic agents with distinct mechanisms of action [EMD 53998 (EMD), which also docks on the myosin molecule, and levosimendan (Levo), which binds to troponin C] were included., Methods: Enzymatically isolated canine cardiomyocytes with intact cell membranes were loaded with Fura-2AM, a Ca 2+ -sensitive, ratiometric, fluorescent dye. Changes in sarcomere length (SL) and intracellular Ca 2+ concentration were recorded in parallel at room temperature, whereas cardiomyocyte contractions were evoked by field stimulation at 0.1 Hz in the presence of different OM, EMD, or Levo concentrations., Results: SL was reduced by about 23% or 9% in the presence of 1 μM OM or 1 μM EMD in the absence of electrical stimulation, whereas 1 μM Levo had no effect on resting SL. Fractional sarcomere shortening was increased by 1 μM EMD or 1 μM Levo to about 152%, but only to about 128% in the presence of 0.03 μM OM. At higher OM concentrations, no significant increase in fractional sarcomere shortening could be recorded. Contraction durations largely increased, whereas the kinetics of contractions and relaxations decreased with increasing OM concentrations. One-micromole EMD or 1 μM Levo had no effects on contraction durations. One-micromole Levo, but not 1 μM EMD, accelerated the kinetics of cardiomyocyte contractions and relaxations. Ca 2+ transient amplitudes were unaffected by all treatments., Conclusions: Our data revealed major distinctions between the cellular effects of myofilament targeted agents (OM, EMD, or Levo) depending on their target proteins and binding sites, although they were compatible with the involvement of Ca 2+ -sensitizing mechanisms for all three drugs. Significant part of the cardiotonic effect of OM relates to the prolongation of systolic contraction in combination with its Ca 2+ -sensitizing effect., (© 2023 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

ABT-333 (dasabuvir) is an antiviral agent used in hepatitis C treatment. The molecule, similarly to some inhibitors of hERG channels, responsible for the delayed rectifier potassium current (I Kr ), contains the methanesulfonamide group. Reduced I Kr current leads to long QT syndrome and early afterdepolarizations (EADs), therefore potentially causing life-threatening arrhythmias and sudden cardiac death. Our goal was to investigate the acute effects of ABT-333 in enzymatically isolated canine left ventricular myocardial cells. Action potentials (APs) and ion currents were recorded with a sharp microelectrode technique and whole-cell patch clamp, respectively. Application of 1 μM ABT-333 prolonged the AP in a reversible manner. The maximal rates of phases 0 and 1 were irreversibly decreased. Higher ABT-333 concentrations caused larger AP prolongation, elevation of the early plateau potential, and reduction of maximal rates of phases 0, 1, and 3. EADs occurred in some cells in 3-30 μM ABT-333 concentrations. The 10 μM ABT-333-sensitive current, recorded with AP voltage clamp, contained a late outward component corresponding to I Kr and an early outward one corresponding to transient outward potassium current (I to ). ABT-333 reduced hERG-channel-mediated ion current in a concentration-dependent, partially reversible manner with a half-inhibitory concentration of 3.2 μM. As the therapeutic plasma concentration of ABT-333 is 1 nM, the arrhythmic risk of ABT-333 is very low, even in the case of drug overdose.

Life-long stable heart function requires a critical balance of intracellular Ca 2+ . Several ion channels and pumps cooperate in a complex machinery that controls the influx, release, and efflux of Ca 2+ . Probably one of the most interesting and most complex players of this crosstalk is the Na + /Ca 2+ exchanger, which represents the main Ca 2+ efflux mechanism; however, under some circumstances, it can also bring Ca 2+ into the cell. Therefore, the inhibition of the Na + /Ca 2+ exchanger has emerged as one of the most promising possible pharmacological targets to increase Ca 2+ levels, to decrease arrhythmogenic depolarizations, and to reduce excessive Ca 2+ influx. In line with this, as a response to increasing demand, several more or less selective Na + /Ca 2+ exchanger inhibitor compounds have been developed. In the past 20 years, several results have been published regarding the effect of Na + /Ca 2+ exchanger inhibition under various circumstances, e.g., species, inhibitor compounds, and experimental conditions; however, the results are often controversial. Does selective Na + /Ca 2+ exchanger inhibition have any future in clinical pharmacological practice? In this review, the experimental results of Na + /Ca 2+ exchanger inhibition are summarized focusing on the data obtained by novel highly selective inhibitors.

TRPV3 (transient receptor potential vanilloid 3) is a pro-inflammatory ion channel mostly expressed by keratinocytes of the human skin. Previous studies have shown that the expression of TRPV3 is markedly upregulated in the lesional epidermis of atopic dermatitis (AD) patients suggesting a potential pathogenetic role of the ion channel in the disease. In the current study, we aimed at defining the molecular and functional expression of TRPV3 in non-lesional skin of AD patients as previous studies implicated that healthy-appearing skin in AD is markedly distinct from normal skin with respect to terminal differentiation and certain immune function abnormalities. By using multiple, complementary immunolabelling and RT-qPCR technologies on full-thickness and epidermal shave biopsy samples from AD patients (lesional, non-lesional) and healthy volunteers, we provide the first evidence that the expression of TRPV3 is markedly upregulated in non-lesional human AD epidermis, similar to lesional AD samples. Of further importance, by using the patch-clamp method on cultured healthy and non-lesional AD keratinocytes, we also show that this upregulation is functional as determined by the significantly augmented TRPV3-specific ion current (induced by agonists) on cultured non-lesional AD keratinocytes when compared to healthy ones., (© 2022 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Cardiac diseases are the leading causes of death, with a growing number of cases worldwide, posing a challenge for both healthcare and research. Therefore, the most relevant aim of cardiac research is to unravel the molecular pathomechanisms and identify new therapeutic targets. Cardiac ryanodine receptor (RyR2), the Ca 2+ release channel of the sarcoplasmic reticulum, is believed to be a good therapeutic target in a group of certain heart diseases, collectively called cardiac ryanopathies. Ryanopathies are associated with the impaired function of the RyR, leading to heart diseases such as congestive heart failure (CHF), catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular dysplasia type 2 (ARVD2), and calcium release deficiency syndrome (CRDS). The aim of the current review is to provide a short insight into the pathological mechanisms of ryanopathies and discuss the pharmacological approaches targeting RyR2.

The patch clamp technique underwent continual advancement and developed numerous variants in cardiac electrophysiology since its introduction in the late 1970s. In the beginning, the capability of the technique was limited to recording one single current from one cell stimulated with a rectangular command pulse. Since that time, the technique has been extended to record multiple currents under various command pulses including action potential. The current review summarizes the development of the patch clamp technique in cardiac electrophysiology with special focus on the potential applications in integrative physiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Horváth, Szentandrássy, Dienes, Kovács, Nánási, Chen-Izu, Izu and Banyasz.)

Late sodium current has long been linked to dysrhythmia and contractile malfunction in the heart. Despite the increasing body of accumulating information on the subject, our understanding of its role in normal or pathologic states is not complete. Even though the role of late sodium current in shaping action potential under physiologic circumstances is debated, it's unquestioned role in arrhythmogenesis keeps it in the focus of research. Transgenic mouse models and isoform-specific pharmacological tools have proved useful in understanding the mechanism of late sodium current in health and disease. This review will outline the mechanism and function of cardiac late sodium current with special focus on the recent advances of the area.

Transient receptor potential melastatin 4 is a unique member of the TRPM protein family and, similarly to TRPM5, is Ca 2+ -sensitive and permeable to monovalent but not divalent cations. It is widely expressed in many organs and is involved in several functions by regulating the membrane potential and Ca 2+ homeostasis in both excitable and non-excitable cells. This part of the review discusses the pharmacological modulation of TRPM4 by listing, comparing, and describing both endogenous and exogenous activators and inhibitors of the ion channel. Moreover, other strategies used to study TRPM4 functions are listed and described. These strategies include siRNA-mediated silencing of TRPM4, dominant-negative TRPM4 variants, and anti-TRPM4 antibodies. TRPM4 is receiving more and more attention and is likely to be the topic of research in the future.

Transient receptor potential melastatin 4 (TRPM4) is a unique member of the TRPM protein family and, similarly to TRPM5, is Ca 2+ sensitive and permeable for monovalent but not divalent cations. It is widely expressed in many organs and is involved in several functions; it regulates membrane potential and Ca 2+ homeostasis in both excitable and non-excitable cells. This part of the review discusses the currently available knowledge about the physiological and pathophysiological roles of TRPM4 in various tissues. These include the physiological functions of TRPM4 in the cells of the Langerhans islets of the pancreas, in various immune functions, in the regulation of vascular tone, in respiratory and other neuronal activities, in chemosensation, and in renal and cardiac physiology. TRPM4 contributes to pathological conditions such as overactive bladder, endothelial dysfunction, various types of malignant diseases and central nervous system conditions including stroke and injuries as well as in cardiac conditions such as arrhythmias, hypertrophy, and ischemia-reperfusion injuries. TRPM4 claims more and more attention and is likely to be the topic of research in the future.

The cardiac late sodium current (I Na,late ) is the small sustained component of the sodium current active during the plateau phase of the action potential. Several studies demonstrated that augmentation of the current can lead to cardiac arrhythmias; therefore, I Na,late is considered as a promising antiarrhythmic target. Fundamentally, enlarged I Na,late increases Na + influx into the cell, which, in turn, is converted to elevated intracellular Ca 2+ concentration through the Na + /Ca 2+ exchanger. The excessive Ca 2+ load is known to be proarrhythmic. This review describes the behavior of the voltage-gated Na + channels generating I Na,late in health and disease and aims to discuss the physiology and pathophysiology of Na + and Ca 2+ homeostasis in context with the enhanced I Na,late demonstrating also the currently accessible antiarrhythmic choices.

Enhancement of the late sodium current (I NaL ) increases arrhythmia propensity in the heart, whereas suppression of the current is antiarrhythmic. In the present study, we investigated I NaL in canine ventricular cardiomyocytes under action potential voltage-clamp conditions using the selective Na + channel inhibitors GS967 and tetrodotoxin. Both 1 µM GS967 and 10 µM tetrodotoxin dissected largely similar inward currents. The amplitude and integral of the GS967-sensitive current was significantly smaller after the reduction of intracellular Ca 2+ concentration ([Ca 2+ ] i ) either by superfusion of the cells with 1 µM nisoldipine or by intracellular application of 10 mM BAPTA. Inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII) by KN-93 or the autocamtide-2-related inhibitor peptide similarly reduced the amplitude and integral of I NaL . Action potential duration was shortened in a reverse rate-dependent manner and the plateau potential was depressed by GS967. This GS967-induced depression of plateau was reduced by pretreatment of the cells with BAPTA-AM. We conclude that (1) I NaL depends on the magnitude of [Ca 2+ ] i in canine ventricular cells, (2) this [Ca 2+ ] i -dependence of I NaL is mediated by the Ca 2+ -dependent activation of CaMKII, and (3) I NaL is augmented by the baseline CaMKII activity.

The profiles of ion currents during the cardiac action potential can be visualized by the action potential voltage clamp technique. To obtain multiple ion current data from the same cell, the "onion peeling" technique, based on sequential pharmacological dissection of ion currents, has to be applied. Combination of the two methods allows recording of several ion current profiles from the same myocyte under largely physiological conditions. Using this approach, we have studied the densities and integrals of the major cardiac inward (I Ca , I NCX , I Na-late ) and outward (I Kr , I Ks , I K1 ) currents in canine ventricular cells and studied the correlation between them. For this purpose, canine ventricular cardiomyocytes were chosen because their electrophysiological properties are similar to those of human ones. Significant positive correlation was observed between the density and integral of I Ca and I Kr , and positive correlation was found also between the integral of I Ca and I NCX . No further correlations were detected. The Ca 2+ -sensitivity of K + currents was studied by comparing their parameters in the case of normal calcium homeostasis and following blockade of I Ca . Out of the three K + currents studied, only I Ks was Ca 2+ -sensitive. The density and integral of I Ks was significantly greater, while its time-to-peak value was shorter at normal Ca 2+ cycling than following I Ca blockade. No differences were detected for I Kr or I K1 in this regard. Present results indicate that the positive correlation between I Ca and I Kr prominently contribute to the balance between inward and outward fluxes during the action potential plateau in canine myocytes. The results also suggest that the profiles of cardiac ion currents have to be studied under physiological conditions, since their behavior may strongly be influenced by the intracellular Ca 2+ homeostasis and the applied membrane potential protocol., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca 2+ -activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca 2+ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca 2+ -activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca 2+ -dependent depolarization of PACs from a resting membrane potential of -44.4 ± 2.9 to -27.7 ± 3 mV. Furthermore, we showed that Ca 2+ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca 2+ transients partially rely on Ca 2+ influx in PACs, the role of TRPM4 was also assessed on Ca 2+ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca 2+ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca 2+ . In conclusion, TRPM4 links intracellular Ca 2+ signaling to membrane potential as a negative feedback regulator of Ca 2+ entry in PACs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75% and 90% of repolarization and decreased the short-term variability of APD 90 . Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (I to ) and late sodium current (I Na,L ) were reduced by approximately 20% and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of I to . The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly I Na,L , therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.

Due to the limited availability of healthy human ventricular tissues, the most suitable animal model has to be applied for electrophysiological and pharmacological studies. This can be best identified by studying the properties of ion currents shaping the action potential in the frequently used laboratory animals, such as dogs, rabbits, guinea pigs, or rats, and comparing them to those of human cardiomyocytes. The authors of this article with the experience of three decades of electrophysiological studies, performed in mammalian and human ventricular tissues and isolated cardiomyocytes, summarize their results obtained regarding the major canine and human cardiac ion currents. Accordingly, L-type Ca 2+ current (I Ca ), late Na + current (I Na-late ), rapid and slow components of the delayed rectifier K + current (I Kr and I Ks , respectively), inward rectifier K + current (I K1 ), transient outward K + current (I to1 ), and Na + /Ca 2+ exchange current (I NCX ) were characterized and compared. Importantly, many of these measurements were performed using the action potential voltage clamp technique allowing for visualization of the actual current profiles flowing during the ventricular action potential. Densities and shapes of these ion currents, as well as the action potential configuration, were similar in human and canine ventricular cells, except for the density of I K1 and the recovery kinetics of I to . I K1 displayed a largely four-fold larger density in canine than human myocytes, and I to recovery from inactivation displayed a somewhat different time course in the two species. On the basis of these results, it is concluded that canine ventricular cells represent a reasonably good model for human myocytes for electrophysiological studies, however, it must be borne in mind that due to their stronger I K1 , the repolarization reserve is more pronounced in canine cells, and moderate differences in the frequency-dependent repolarization patterns can also be anticipated.

Enhancement of the late Na + current (I NaL ) increases arrhythmia propensity in the heart, while suppression of the current is antiarrhythmic. GS967 is an agent considered as a selective blocker of I NaL . In the present study, effects of GS967 on I NaL and action potential (AP) morphology were studied in canine ventricular myocytes by using conventional voltage clamp, action potential voltage clamp and sharp microelectrode techniques. The effects of GS967 (1 µM) were compared to those of the class I/B antiarrhythmic compound mexiletine (40 µM). Under conventional voltage clamp conditions, I NaL was significantly suppressed by GS967 and mexiletine, causing 80.4 ± 2.2% and 59.1 ± 1.8% reduction of the densities of I NaL measured at 50 ms of depolarization, and 79.0 ± 3.1% and 63.3 ± 2.7% reduction of the corresponding current integrals, respectively. Both drugs shifted the voltage dependence of the steady-state inactivation curve of I NaL towards negative potentials. GS967 and mexiletine dissected inward I NaL profiles under AP voltage clamp conditions having densities, measured at 50% of AP duration (APD), of -0.37 ± 0.07 and -0.28 ± 0.03 A/F, and current integrals of -56.7 ± 9.1 and -46.6 ± 5.5 mC/F, respectively. Drug effects on peak Na + current (I NaP ) were assessed by recording the maximum velocity of AP upstroke (V + max ) in multicellular preparations. The offset time constant was threefold faster for GS967 than mexiletine (110 ms versus 289 ms), while the onset of the rate-dependent block was slower in the case of GS967. Effects on beat-to-beat variability of APD was studied in isolated myocytes. Beat-to-beat variability was significantly decreased by both GS967 and mexiletine (reduction of 42.1 ± 6.5% and 24.6 ± 12.8%, respectively) while their shortening effect on APD was comparable. It is concluded that the electrophysiological effects of GS967 are similar to those of mexiletine, but with somewhat faster offset kinetics of V + max block. However, since GS967 depressed V + max and I NaL at the same concentration, the current view that GS967 represents a new class of drugs that selectively block I NaL has to be questioned and it is suggested that GS967 should be classified as a class I/B antiarrhythmic agent.

It has long been known that the electrophysiological effects of many cardioactive drugs strongly depend on the rate dependent frequency. This was recognized first for class I antiarrhythmic agents: their V max suppressive effect was attenuated at long cycle lengths. Later many Ca 2+ channel blockers were also found to follow such kinetics. The explanation was provided by the modulated and the guarded receptor theories. Regarding the duration of cardiac action potentials (APD) an opposite frequency-dependence was observed, i.e. the drug-induced changes in APD were proportional with the cycle length of stimulation, therefore it was referred as "reverse rate-dependency". The beat-to-beat, or short term variability of APD (SV) has been recognized as an important proarrhythmic mechanism, its magnitude can be used as an arrhythmia predictor. SV is modulated by several cardioactive agents, however, these drugs modify also APD itself. In order to clear the drug-specific effects on SV from the concomitant unspecific APD-change related ones, the term of "relative variability" was introduced. Relative variability is increased by ion channel blockers that decrease the negative feedback control of APD (i.e. blockers of I Ca , I Kr and I Ks ) and also by elevation of cytosolic Ca 2+ . Cardiac arrhythmias are also often categorized according to the characteristic heart rate (tachy- and bradyarrhythmias). Tachycardia is proarrhythmic primarily due to the concomitant Ca 2+ overload causing delayed afterdepolarizations. Early afterdepolarizations (EADs) are complications of the bradycardic heart. What is common in the reverse rate-dependent nature of drug action on APD, increased SV and EAD incidence associated with bradycardia., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Based on recent findings, an increased late sodium current (I Na,late ) plays an important pathophysiological role in cardiac diseases, including rhythm disorders. The article first describes what is I Na,late and how it functions under physiological circumstances. Next, it shows the wide range of cellular mechanisms that can contribute to an increased I Na,late in heart diseases, and also discusses how the upregulated I Na,late can play a role in the generation of cardiac arrhythmias. The last part of the article is about I Na,late inhibiting drugs as potential antiarrhythmic agents, based on experimental and preclinical data as well as in the light of clinical trials., (Copyright © 2020 Horváth, Hézső, Kiss, Kistamás, Magyar, Nánási and Bányász.)

Calcium ions (Ca 2+ ) play a major role in the cardiac excitation-contraction coupling. Intracellular Ca 2+ concentration increases during systole and falls in diastole thereby determining cardiac contraction and relaxation. Normal cardiac function also requires perfect organization of the ion currents at the cellular level to drive action potentials and to maintain action potential propagation and electrical homogeneity at the tissue level. Any imbalance in Ca 2+ homeostasis of a cardiac myocyte can lead to electrical disturbances. This review aims to discuss cardiac physiology and pathophysiology from the elementary membrane processes that can cause the electrical instability of the ventricular myocytes through intracellular Ca 2+ handling maladies to inherited and acquired arrhythmias. Finally, the paper will discuss the current therapeutic approaches targeting cardiac arrhythmias., (Copyright © 2020 Kistamás, Veress, Horváth, Bányász, Nánási and Eisner.)

Although late sodium current (I Na-late ) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of I Na-late in canine and guinea pig ventricular cells and compare them to I Na-late recorded in undiseased human hearts. I Na-late was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic- or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human I Na-late monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine I Na-late could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo I Na-late profile in guinea pig was due to the slower decay of I Na-late in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of I Na-late . At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of I Na-late . These results should be taken into account when pharmacological studies with I Na-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with I Na-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of I Na-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ventricular fibrillation (VF) and sudden cardiac death (SCD) are predominantly caused by channelopathies and cardiomyopathies in youngsters and coronary heart disease in the elderly. Temporary factors, e.g., electrolyte imbalance, drug interactions, and substance abuses may play an additive role in arrhythmogenesis. Ectopic automaticity, triggered activity, and reentry mechanisms are known as important electrophysiological substrates for VF determining the antiarrhythmic therapies at the same time. Emergency need for electrical cardioversion is supported by the fact that every minute without defibrillation decreases survival rates by approximately 7%-10%. Thus, early defibrillation is an essential part of antiarrhythmic emergency management. Drug therapy has its relevance rather in the prevention of sudden cardiac death, where early recognition and treatment of the underlying disease has significant importance. Cardioprotective and antiarrhythmic effects of beta blockers in patients predisposed to sudden cardiac death were highlighted in numerous studies, hence nowadays these drugs are considered to be the cornerstones of the prevention and treatment of life-threatening ventricular arrhythmias. Nevertheless, other medical therapies have not been proven to be useful in the prevention of VF. Although amiodarone has shown positive results occasionally, this was not demonstrated to be consistent. Furthermore, the potential proarrhythmic effects of drugs may also limit their applicability. Based on these unfavorable observations we highlight the importance of arrhythmia prevention, where echocardiography, electrocardiography and laboratory testing play a significant role even in the emergency setting. In the following we provide a summary on the latest developments on cardiopulmonary resuscitation, and the evaluation and preventive treatment possibilities of patients with increased susceptibility to VF and SCD., (Copyright © 2020 Szabó, Ujvárosy, Ötvös, Sebestyén and Nánási.)

Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial results showing that dantrolene inhibited Ca 2+ release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single RyR channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene's action was lost during the purification procedure of RyR. Recently, Mg 2+ was demonstrated to be the essential factor for dantrolene to inhibit Ca 2+ release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca 2+ release and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca 2+ release experiments demonstrated that the effect of dantrolene and Mg 2+ was cooperative and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 µ M dantrolene reduced RyR channel open probability by ∼50% in the presence of 3 mM free Mg 2+ and 1 mM ATP, whereas channel activity further decreased to ∼20% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that supports the direct, Mg 2+ -dependent mechanism of dantrolene's action and suggests that dantrolene also requires ATP to inhibit RyR., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The big conductance Ca 2+ -dependent K + channel, also known as BK, MaxiK, Slo1, or KCa1.1, is a ligand- and voltage-gated K + channel. Although structure-function studies of the past decades, involving mutagenesis and electrophysiological measurements, revealed fine details of the mechanism of BK channel gating, the exact molecular details remained unknown until the quaternary structure of the protein has been solved at a resolution of 3.5 Å using cryo-electron microscopy. In this short review, we are going to summarize these results and interpret the gating model of the BK channel in the light of the recent structural results.

Purpose: Primary fluid secretion in secretory epithelia relies on the unidirectional transport of ions and water across a single cell layer. This mechanism requires the asymmetric apico-basal distribution of ion transporters and intracellular Ca 2+ signaling. The primary aim of the present study was to verify the localization and the identity of Ca 2+ -dependent ion channels in acinar cells of the mouse lacrimal gland., Methods: Whole-cell patch-clamp-electrophysiology, spatially localized flash-photolysis of Ca 2+ and temporally resolved digital Ca 2+ -imaging was combined. Immunostaining of enzymatically isolated mouse lacrimal acinar cells was performed., Results: We show that the Ca 2+ -dependent K + -conductance is paxilline-sensitive, abundant in the luminal, but negligible in the basal membrane; and co-localizes with Cl - -conductance. These data suggest that both Cl - and K + are secreted into the lumen and thus they account for the high luminal [Cl - ] (∼141 mM), but not for the relatively low [K + ] (<17 mM) of the primary fluid. Accordingly, these results also imply that K + must be reabsorbed from the primary tear fluid by the acinar cells. We hypothesized that apically-localized Na + -K + pumps are responsible for K + -reabsorption. To test this possibility, immunostaining of lacrimal acinar cells was performed using anti-Na + -K + ATP-ase antibody. We found positive fluorescence signal not only in the basal, but in the apical membrane of acinar cells too., Conclusions: Based on these results we propose a new primary fluid-secretion model in the lacrimal gland, in which the paracellular pathway of Na + secretion is supplemented by a transcellular pathway driven by apical Na + -K + pumps., (Copyright © 2019. Published by Elsevier Inc.)

The role of transient receptor potential melastatin 4 (TRPM4) channels has been frequently tested using their inhibitor 9-phenanthrol in various cardiac preparations; however, the selectivity of the compound is uncertain. Therefore, in the present study, the concentration-dependent effects of 9-phenanthrol on major ionic currents were studied in canine isolated ventricular cells using whole-cell configuration of the patch-clamp technique and 10 mM BAPTA-containing pipette solution to prevent the Ca 2+ -dependent activation of TRPM4 channels. Transient outward (I to1 ), rapid delayed rectifier (I Kr ), and inward rectifier (I K1 ) K + currents were suppressed by 10 and 30 μM 9-phenanthrol with the blocking potency for I K1 < I Kr < I to1 and partial reversibility. L-type Ca 2+ current was not affected up to the concentration of 30 μM. In addition, a steady outward current was detected at voltages positive to -40 mV in 9-phenanthrol, which was larger at more positive voltages and larger 9-phenanthrol concentrations. Action potentials were recorded using microelectrodes. Maximal rate of depolarization, phase-1 repolarization, and terminal repolarization were decreased and the plateau potential was depressed by 9-phenanthrol (3-30 μM), congruently with the observed alterations of ionic currents. Significant action potential prolongation was observed by 9-phenanthrol in the majority of the studied cells, but only at 30 μM concentration. In conclusion, 9-phenanthrol is not selective to TRPM4 channels in canine ventricular myocardium; therefore, its application as a TRPM4 blocker can be appropriate only in expression systems but not in native cardiac cells.

[This corrects the article DOI: 10.1371/journal.pone.0151461.].

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl - efflux and the subsequent paracellular Na + transport. In this model, the Na + -K + pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl - transport via basolateral Na + -K + -2Cl - cotransport is generated by the Na + -K + pump. In addition, the continuous electrochemical gradient for Cl - flow during acinar cell stimulation is maintained by the basolateral K + efflux. However, using a combination of single-cell electrophysiology and Ca 2+ -imaging, we demonstrate that photolysis of Ca 2+ close to the apical membrane of parotid acinar cells triggered significant K + current, indicating that a substantial amount of K + is secreted into the lumen during stimulation. Nevertheless, the K + content of the primary saliva is relatively low, suggesting that K + might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na + -K + pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K + from and secretes Na + to the lumen, which can partially supplement the paracellular Na + pathway.

Transient receptor potential (TRP) ion channels were first characterized on neurons, where they are classically implicated in sensory functions; however, research in recent decades has shown that many of these channels are also expressed on nonneuronal cell types. Emerging findings have highlighted the role of TRP channels in the skin, where they have been shown to be important in numerous cutaneous functions. Of particular interest is TRPV3, which was first described on keratinocytes. Its functional importance was supported when its gain-of-function mutation was linked to Olmsted syndrome, which is characterized by palmoplantar keratoderma, periorifacial hyperkeratosis, diffuse hypotrichosis and alopecia, and itch. Despite these exciting results, we have no information about the role and functionality of TRPV3 on keratinocytes at the cellular level. In this study, we identified TRPV3 expression both on human skin and cultured epidermal keratinocytes. TRPV3 stimulation was found to function as a Ca 2+ -permeable ion channel that suppresses proliferation of epidermal keratinocytes and induces cell death. Stimulation of the channel also triggers a strong proinflammatory response via the NF-κB pathway. Collectively, our data show that TRPV3 is functionally expressed on human epidermal keratinocytes and that it plays a role in cutaneous inflammatory processes., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Na + /Ca 2+ exchanger (NCX) is the main Ca 2+ transporter in cardiac myocytes. Its inhibition could be expected to exert positive inotropic action by accumulation of cytosolic Ca 2+ ([Ca 2+ ] i ). However, we have observed only a marginal positive inotropic effect upon selective inhibition of NCX, which was enhanced when forward activity was facilitated. Here we attempted to clarify the underlying mechanism of the limited inotropic action of selective NCX inhibition by a novel inhibitor ORM-10962 on canine ventricular myocytes. 1µM ORM-10962 reduced the Ca 2+ content of sarcoplasmic reticulum (SR) when the reverse NCX was favoured, while SR Ca 2+ content was increased by ORM-10962 under conditions favouring the forward activity, like elevation of [Ca 2+ ] i . L-type Ca 2+ current (I Ca ) was not affected by 1µM ORM-10962 in the absence of SR Ca 2+ release, while I Ca was suppressed by ORM-10962 during normal Ca 2+ cycling. The apparent degree of forward NCX inhibition was dependent on the elevation of [Ca 2+ ] i , suggesting that an increased driving force of forward NCX can also limit the accumulation of [Ca 2+ i ]. We concluded that in healthy myocardium the possible positive inotropic potential of NCX inhibition is considerably weaker than it was expected earlier by theoretical assumptions. The underlying mechanism may involve the autoregulation of Ca 2+ handling and/or the preserved inducibility of forward NCX by high [Ca 2+ ] i . This limitation of selective NCX inhibition seen in undiseased myocardium requires further studies in failing heart, which may allow correct evaluation of the potential therapeutic value of selective NCX inhibitors in the treatment of heart failure., (Copyright © 2017. Published by Elsevier B.V.)

Omecamtiv mecarbil (OM) is a myosin activator agent developed for the treatment of heart failure. OM was reported to increase left ventricular ejection fraction and systolic ejection time, but little is known about the effect of heart rate on the action of OM. The present study, therefore, was designed to investigate the effects of OM on unloaded cell shortening and intracellular Ca 2+ ([Ca 2+ ] i ) transients as a function of the pacing frequency. Isolated cardiomyocytes were stimulated at various frequencies under steady-state conditions. Cell length was monitored by an optical edge detector and changes in [Ca 2+ ] i were followed using the Ca 2+ -sensitive dye Fura-2. At the pacing frequency of 1 Hz, OM (1-10 μM) significantly decreased both diastolic and systolic cell length, however, fractional shortening was augmented only by 1 μM OM. Time to peak tension and time of 90% relaxation were progressively increased by OM. At the frequency of 2 Hz, diastolic cell length was reduced by 10 μM OM to a larger extent than systolic cell length, resulting in a significantly decreased fractional shortening under these conditions. OM had no effect on the parameters of the [Ca 2+ ] i transient at any pacing frequency. The results suggest that supratherapeutic concentrations of OM may decrease rather than increase the force of cardiac contraction especially in tachycardic patients.

Beat-to-beat variability of cardiac action potential duration (short-term variability, SV) is a common feature of various cardiac preparations, including the human heart. Although it is believed to be one of the best arrhythmia predictors, the underlying mechanisms are not fully understood at present. The magnitude of SV is basically determined by the intensity of cell-to-cell coupling in multicellular preparations and by the duration of the action potential (APD). To compensate for the APD-dependent nature of SV, the concept of relative SV (RSV) has been introduced by normalizing the changes of SV to the concomitant changes in APD. RSV is reduced by I Ca , I Kr , and I Ks while increased by I Na , suggesting that ion currents involved in the negative feedback regulation of APD tend to keep RSV at a low level. RSV is also influenced by intracellular calcium concentration and tissue redox potential. The clinical implications of APD variability is discussed in detail.

The role of Ca 2+ -activated Cl - current (I Cl(Ca) ) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca 2+ -overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by I Cl(Ca) . Therefore our goal was to examine the role of I Cl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. I Cl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating I Cl(Ca) , was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither I Cl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of I Cl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting I Cl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for I Cl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: In this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined., Methods: Ion currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 μg/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts., Results: ORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 μM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 μM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts., Conclusions: The present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain., Competing Interests: T. Koskaleinen, J. Levijoki, L. Otsomaa and P. Pollesello, employed by Orion Pharma, have been involved in the development of ORM-10962. Other authors have nothing to declare. The commercial adherence of the Orion Pharma does not alter our adherence to PLOS ONE policies on sharing data and materials.

Ca(2+)-activated Cl(-) current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Abnormal Ca(2+)homeostasis is often associated with chronic cardiovascular diseases, such as hypertension, heart failure or cardiac arrhythmias, and typically contributes to the basic ethiology of the disease. Pharmacological targeting of cardiac Ca(2+)handling has great therapeutic potential offering invaluable options for the prevention, slowing down the progression or suppression of the harmful outcomes like life threatening cardiac arrhythmias. In this review we outline the existing knowledge on the involvement of malfunction of the ryanodine receptor and the Na(+)-Ca(2+)exchanger in disturbances of Ca(2+)homeostasis and discuss important proof of concept pharmacological studies targeting these mechanisms in context of hypertension, heart failure, atrial fibrillation and ventricular arrhythmias. We emphasize the promising results of preclinical studies underpinning the potential benefits of the therapeutic strategies based on ryanodine receptor or Na(+)-Ca(2+)exchanger inhibition., (Copyright © 2016 Elsevier B.V. All rights reserved.)